Fluorescence-based molecular Imaging in small animals is having a major impact on drug development and disease research. However, a significant challenge to imaging targeted fluorescent markers in vivo remains: unless the labeled regions are located superficially; localization, quantitation and host organ identification are impeded by the effects of light scattering and absorption. Orthotopic tumor and disease models are increasingly preferred over less biologically relevant subcutaneous xenografts. In such studies, substantial difficulties are encountered in longitudinal studies where animals are growing and are positioned differently for each measurement. We believe that a single imaging advance could address many of these issues, and advance the utility of in-vivo molecular imaging: an exact anatomical co-registration technique that does not rely on multimodal techniques. This proposal describes dynamic molecular imaging (DMI), an approach that can provide co-registered anatomical information by exploiting in-vivo pharmacokinetics of dyes in small animals in a simple and inexpensive way. We demonstrate that by acquiring a time-series of optical images during injection of an inert dye, we can repeatably and accurately delineate the major internal organs of mice using optical imaging alone. This is possible because each major organ is "illuminated" by the kinetics of dye passing through it in such a manner as to make it distinguishable from other structures. Spatiotemporal analysis can exploit these characteristic time courses to allow the body-surface representation of each organ to be visualized. These in- vivo anatomical maps can be overlaid onto simultaneously acquired images of a targeted molecular probe (detected and distinguished from the mapping dye via multispectral imaging techniques, if necessary) to significantly aid in identification of the probe's anatomical and physical location. Using CRi's existing and prototype 2D, "2.5D" and true 3D multispectral mouse imaging systems, we propose to test and refine a DMI approach. Based on our findings to date, we will examine and exploit in-vivo pharmacokinetics of the near-infrared dye, indocyanine green, to generate delineated surface projections of individual organs. Co-registering this surface map with surface projections of detected targeted labels will allow the targeted probe's 3D spatial location to be inferred. This information can further be used to improve quantitative accuracy in longitudinal molecular imaging studies of deep targets. [unreadable] [unreadable] [unreadable]